Physiologic Effects of Housing Rats in Metabolic Cages.

Currently, metabolic cages (MC) are the only way to achieve serial sampling of urine and feces in rodents. However, the use of this caging creates a dramatic change from an animal's usual microenvironment. Here we sought to examine the effect of MC on physiologic parameters that are stress-responsive in rats. We surgically implanted 8 male Wistar rats (weight, 150 to 175 g) with telemetric transmitters and allowed them to recover for at least 2 wk. At the beginning of the study, the rats were moved to conventional open-top cages, and telemetry recording was initiated. After 24 h, the rats were moved to MC or to another conventional cage and the recording continued for another 24 h. Finally, the rats were returned to their home cages, and telemetry recording was performed for a final 24 h. After 10 days, this process was then repeated, with MC and conventional assignments switched. During the 78-h monitoring period, we recorded heart rate, arterial blood pressure, locomotor activity, body weight, and food and water consumption. Heart rate and arterial blood pressure showed transient but significant changes. Locomotor activity during the dark phase was greatly decreased in MC compared with conventional cages, perhaps due to space constraints. In addition, when the rats were housed in MC, they showed a small but significant weight loss. Food consumption did not differ between housing environments, but water consumption was lower when rats were in MC. In conclusion, the housing of rats in MC for 24 h can elicit mild and reversible cardiovascular changes. This finding is consistent with European Directive 2010/63/EU, which considers short-term (less than 24 h) restraint in MC a procedure of mild severity.

Pharmacological Profile of AZD8871 (LAS191351), a Novel Inhaled Dual M3 Receptor Antagonist/ß 2-Adrenoceptor Agonist Molecule with Long-Lasting Effects and Favorable Safety Profile.

AZD8871 is a novel muscarinic antagonist and ß 2-adrenoceptor agonist in development for chronic obstructive pulmonary disease. This study describes the pharmacological profile of AZD8871 in in vitro and in vivo assays. AZD8871 is potent at the human M3 receptor (pIC50 in binding assays: 9.5) and shows kinetic selectivity for the M3 (half-life: 4.97 hours) over the M2 receptor (half-life: 0.46 hour). It is selective for the ß 2-adrenoceptor over the ß 1 and ß 3 subtypes (3- and 6-fold, respectively) and shows dual antimuscarinic and ß 2-adrenoceptor functional activity in isolated guinea pig tissue (pIC50 in electrically stimulated trachea: 8.6; pEC50 in spontaneous tone isolated trachea: 8.8, respectively), which are sustained over time. AZD8871 exhibits a higher muscarinic component than batefenterol in human bronchi, with a shift in potency under propranolol blockade of 2- and 6-fold, respectively, together with a persisting relaxation (5.3% recovery at 8 hours). Nebulized AZD8871 prevents acetylcholine-induced bronchoconstriction in both guinea pig and dog with minimal effects on salivation and heart rate at doses with bronchoprotective activity. Moreover, AZD8871 shows long-lasting effects in dog, with a bronchoprotective half-life longer than 24 hours. In conclusion, these studies demonstrate that AZD8871 is a dual-acting molecule with a high muscarinic component and a long residence time at the M3 receptor; moreover, its preclinical profile in animal models suggests a once-daily dosing in humans and a favorable safety profile. Thus, AZD8871 has the potential to be a next generation of inhaled bronchodilators in respiratory diseases.

4-Amino-7,8-dihydro-1,6-naphthyridin-5(6 H)-ones as Inhaled Phosphodiesterase Type 4 (PDE4) Inhibitors: Structural Biology and Structure-Activity Relationships.

Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with subnanomolar enzymatic potencies. X-ray crystallography confirmed the binding mode of this novel template. We achieved compounds with double-digit picomolar enzymatic potencies through further structure-based design by targeting both the PDE4 enzyme metal-binding pocket and occupying the solvent-filled pocket. A strategy for lung retention and long duration of action based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung neutrophilia model by suspension microspray and dry powder administration. Suspension microspray of potent compounds showed in vivo efficacy with a clear dose-response. Despite sustained lung levels, dry powder administration performed much less well and without proper dose-response, highlighting clear differences between the two formulations. This indicates a deficiency in the low aqueous solubility strategy for long duration lung efficacy.

Abediterol (LAS100977), an inhaled long-acting ß2-adrenoceptor agonist, has a fast association rate and long residence time at receptor.

This study describes the association rate and residence time of abediterol, a novel long-acting ß2-adrenoceptor agonist (LABA) in Phase II development for treatment of asthma and COPD, in comparison with indacaterol, olodaterol, vilanterol and salmeterol, for both human ß1- and ß2-adrenoceptors. Abediterol association and dissociation rates were monitored directly by using its tritiated form. Moreover, association was determined indirectly using experimental Ki and koff obtained from assays performed with unlabelled compound. Dissociation was also studied indirectly by measuring the association rate of 3H-CGP12177 to beta adrenoceptors previously occupied by unlabelled compounds. Abediterol shows a fast association for the ß2-adrenoceptor (kon 1.4 × 107 ± 1.8 × 106M-1min-1) while its dissociation rate is between 30 and 64 times slower than that of the reference LABA compounds tested, with a residence time of 91.3 ± 13.3min (measured directly) and 185.5 ± 7.5min (measured indirectly). Abediterol shows kinetic selectivity for the ß2- over the ß1-adrenoceptor, with a dissociation rate from the ß1-adrenoceptor similar to the other LABA compounds tested. In conclusion, abediterol is a potent LABA with a fast association rate and a long residence time at ß2-adrenoceptors. These data are in agreement with the onset and duration of action of abediterol shown in humans.

Pharmacological preclinical characterization of LAS190792, a novel inhaled bifunctional muscarinic receptor antagonist /ß2-adrenoceptor agonist (MABA) molecule.

LAS190792 is a novel muscarinic antagonist and ß2-adrenoceptor agonist in development for chronic respiratory diseases. This study investigated the pharmacological profile of LAS190792 in comparison to batefenterol, tiotropium, indacaterol and olodaterol. LAS190792 is potent at the human M3 receptor (pIC50: 8.8 in binding assays). It is selective for the ß2-adrenoceptor over the ß1-and ß3-adrenoceptor, and shows a functional potency in a similar range to batefenterol and LABA compounds (pEC50 in spontaneous tone isolated trachea: 9.6). The relaxant potency of LAS190792 in electrically stimulated tissue is similar to batefenterol, with an antimuscarinic activity in presence of propranolol slightly higher than batefenterol (pIC50 of 8.3 versus 7.9 in human tissue). LAS190792 exhibits a sustained duration of action in isolated tissue longer than that of batefenterol. Nebulized LAS190792 inhibits acetylcholine-induced bronchoconstriction in dog with minimal cardiac effects and sustained bronchodilation (t1/2: 13.3 h). In conclusion, these studies suggest that LAS190792 is a dual-acting muscarinic antagonist ß2-adrenoceptor agonist that has the potential to be a next generation bronchodilator with long-lasting effects and wide safety margin in humans.

Characterization of the chloroquine-induced mouse model of pruritus using an automated behavioural system.

Pruritus is a major symptom of several dermatological diseases but has limited therapeutic options available. Animal models replicating the pathophysiology of pruritus are needed to support the development of new drugs. Induction of pruritus by chloroquine (CQ) in mice is widely used, although, as with similar models, it has low throughput and does not distinguish between antipruritic effects and confounding factors such as sedation. To overcome these issues, we incorporated into the model an automated system that measures both scratching and locomotor behaviour simultaneously. We combined this system with the determination of CQ levels in different tissues to understand the impact of the route of CQ administration on the pruritogenic response. We concluded that whereas oral CQ does not induce pruritus due to insufficient skin levels, the bell-shaped curve of pruritus observed following subcutaneous administration is due to toxicity at high doses. We validated the model with several drugs currently used in humans: nalfurafine, aprepitant, cyproheptadine and amitriptyline. By comparing the effects of the drugs on both scratching and locomotor activity, we concluded that nalfurafine and aprepitant can exhibit efficacy at doses devoid of central effects, whereas central effects drove the efficacy of the other two drugs. This was further confirmed using non-brain-penetrant drugs. Moreover, as anticipated, anti-inflammatory drugs showed no efficacy. In conclusion, the use of an automated integrated behavioural assessment in CQ-induced pruritus makes the assay suitable for screening purposes and allows for a correct interpretation of the antipruritic effect of the compounds evaluated.

Biphenyl Pyridazinone Derivatives as Inhaled PDE4 Inhibitors: Structural Biology and Structure-Activity Relationships.

Cyclic nucleotide cAMP is a ubiquitous secondary messenger involved in a plethora of cellular responses to biological agents involving activation of adenylyl cyclase. Its intracellular levels are tightly controlled by a family of cyclic nucleotide degrading enzymes, the PDEs. In recent years, cyclic nucleotide phosphodiesterase type 4 (PDE4) has aroused scientific attention as a suitable target for anti-inflammatory therapy in respiratory diseases, particularly in the management of asthma and COPD. Here we describe our efforts to discover novel, highly potent inhaled inhibitors of PDE4. Through structure based design, with the inclusion of a variety of functional groups and physicochemical profiles in order to occupy the solvent-filled pocket of the PDE4 enzyme, we modified the structure of our oral PDE4 inhibitors to reach compounds down to picomolar enzymatic potencies while at the same time tackling successfully an uncovered selectivity issue with the adenosine receptors. In vitro potencies were demonstrated in a rat lung neutrophilia model by administration of a suspension with a Penn-Century MicroSprayer Aerosolizer.

Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice.

BACKGROUND AND PURPOSE: Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. METHODS: Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. RESULTS: In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. CONCLUSIONS: The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.

Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients.

BACKGROUND: Inhaled corticosteroid (ICS) with long-acting beta-2 agonists is a well-documented combination therapy for chronic obstructive pulmonary disease (COPD) based on its additive anti-inflammatory properties. By contrast, the recommendation of ICS in combination with long-acting muscarinic antagonist (LAMA) is not evidence-based. In this study, neutrophils obtained from COPD patients were used to compare the anti-inflammatory effects of aclidinium bromide (a long-acting muscarinic antagonist) with corticosteroids and their potential additive effect. METHODS: Human sputum and blood neutrophils were isolated from healthy individuals (n = 37), patients with stable COPD (n = 52) and those with exacerbated COPD (n = 16). The cells were incubated with corticosteroid fluticasone propionate (0.1 nM-1 µM), aclidinium bromide (0.1 nM-1 µM) or a combination thereof and stimulated with 1 µg of lipopolysaccharide/ml or 5 % cigarette smoke extract. Levels of the pro-inflammatory mediators interleukin-8, matrix metalloproteinase-9, CCL-5, granulocyte-macrophage colony-stimulating factor and interleukin-1ß were measured and the mechanisms of corticosteroid resistance evaluated at the end of the incubation. RESULTS: The non-neuronal cholinergic system was over-expressed in neutrophils from COPD patients, as evidenced by increases in the expression of muscarinic receptors (M2, M4 and M5), choline acetyltransferase and vesicular acetylcholine transporter. Aclidinium bromide demonstrated anti-inflammatory effects on neutrophils from COPD patients, reversing their resistance to corticosteroids. Additive effects of combined aclidinium bromide and fluticasone propionate in blocking M2 receptor levels, inhibiting phosphoinositide 3-kinase-δ and enhancing the glucocorticoid response element transcription factor were demonstrated and were accompanied by an increase in the corticosteroid-induced expression of anti-inflammatory-related genes. CONCLUSIONS: LAMAs potentiate the anti-inflammatory effects of corticosteroids in neutrophils from COPD patients in vitro, thus providing a scientific rationale for their use in combination with corticosteroids in the treatment of COPD.

In vitro and in vivo preclinical profile of abediterol (LAS100977), an inhaled long-acting ß2-adrenoceptor agonist, compared with indacaterol, olodaterol and vilanterol.

Abediterol is a novel long-acting ß2-adrenoceptor agonist (LABA) currently in development for once-daily combination maintenance therapy of asthma and COPD. This study investigated the preclinical profile of abediterol in terms of affinity, potency, selectivity, duration of action and cardiac effects in comparison to the marketed once-daily LABAs indacaterol, olodaterol and vilanterol. Abediterol was the compound with the highest in vitro potency for dog, guinea pig and human ß2-adrenoceptors. In electrical field stimulated guinea pig trachea, abediterol demonstrated 5-, 44- and 77-fold greater potency than olodaterol, indacaterol and vilanterol, respectively. In anaesthetised guinea pigs, inhaled abediterol was also the most potent compound, with 5-20 times higher bronchoprotective potency than other once-daily LABAs against acetylcholine. The bronchoprotective half-life of abediterol in guinea pigs was 36h compared with 51h for indacaterol, 47h for olodaterol, and 18h for vilanterol. In anaesthetised dogs, abediterol also inhibited acetylcholine-induced bronchoconstriction, with higher potency than olodaterol and vilanterol [ID40 (dose inhibiting bronchoconstriction by 40%) of 0.059µg/kg, 0.180µg/kg and 2.870µg/kg, respectively]. In parallel, effects on heart rate in dogs were also measured. Abediterol showed greater safety index (defined as the ratio of the maximal dose without effect on heart rate and the ID40) than olodaterol and vilanterol (10.5 versus 4.9 and 2.4, respectively). Taken together, these data suggest that abediterol offers potent bronchodilation and a sustained duration of action suited to once-daily dosing, plus a reduced potential for class-related cardiac side effects.

Effects of Changing to Individually Ventilated Caging on Guinea Pigs (Cavia porcellus).

The goal of this study was to evaluate the effect of changing to IVC housing on guinea pigs by recording several physiologic parameters in guinea pigs housed sequentially in open-top cages (OTC) and IVC. To register heart rate and locomotor activity, 10 male Dunkin-Hartley guinea pigs implanted with telemetric transmitters were moved from OTC to new, freshly prepared OTC or IVC and subsequently monitored by telemetry during the 4 d after the first cage change. Body weight and food consumption were measured twice during the study. Comparison of data from OTC- and IVC-housed guinea pigs showed no relevant differences in heart rate (mean ± 1 SD; 213 ± 10 bpm and 207 ± 9 bpm, respectively) at any time point. In contrast, locomotor activity varied: whereas activity during the first 4 h after the change of cage type was greater in IVC-housed animals, that during the following 24 h was greater in OTC but was similar between groups thereafter. Animals housed in OTC consumed more food than did those in IVC and, under both conditions, consumption was statistically related to body weight changes. Together, these results show that a change to IVC housing induced only transient increases in locomotor activity in guinea pigs without a marked increase in heart rate but with a decrease in food consumption. Because decreased food consumption was the only stress-associated sign during the 4-d observation, longer studies are needed to ascertain the importance of this finding.

Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides as potent and long acting muscarinic antagonists.

Novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides have been identified as potent M3 muscarinic antagonists with a long duration of action in an in vivo model of bronchoconstriction. The synthesis, structure-activity relationships and biological evaluation of this series of compounds are reported.

Pharmacological characterization of the interaction between aclidinium bromide and formoterol fumarate on human isolated bronchi.

Long-acting muscarinic receptor antagonists (LAMAs) and long-acting ß2-adrenoceptor agonists (LABAs) cause airway smooth muscle (ASM) relaxation via different signal transduction pathways, but there are limited data concerning the interaction between these two drug classes on human bronchi. The aim of this study was to investigate the potential synergistic interaction between aclidinium bromide and formoterol fumarate on the relaxation of human ASM. We evaluated the influence of aclidinium bromide and formoterol fumarate on the contractile response induced by acetylcholine or electrical field stimulation (EFS) on human isolated airways (segmental bronchi and bronchioles). We analyzed the potential synergistic interaction between the compounds when administered in combination by using Bliss independence (BI) theory. Both aclidinium bromide and formoterol fumarate completely relaxed segmental bronchi pre-contracted with acetylcholine (Emax: 97.5±2.6% and 96.4±1.1%; pEC50 8.5±0.1 and 8.8±0.1; respectively). Formoterol fumarate, but not aclidinium bromide, abolished the contraction induced by acetylcholine in bronchioles (Emax: 68.1±4.5% and 99.0±5.6%; pEC50 7.9±0.3 and 8.4±0.3; respectively). The BI analysis indicated synergistic interaction at low concentrations in segmental bronchi (+18.4±2.7%; P<0.05 versus expected effect) and from low to high concentrations in bronchioles (+19.7±0.9%; P<0.05 versus expected effect). Low concentrations of both drugs produced a synergistic relaxant interaction on isolated bronchi stimulated with EFS that was sustained for 6h post-treatment (+55.1±9.4%; P<0.05 versus expected effect). These results suggest that combining aclidinium bromide plus formoterol fumarate provides synergistic benefit on ASM relaxation of both medium and small human airways, which may have major implications for the use of this combination in the clinic.

The in vitro and in vivo profile of aclidinium bromide in comparison with glycopyrronium bromide.

This study characterised the in vitro and in vivo profiles of two novel long-acting muscarinic antagonists, aclidinium bromide and glycopyrronium bromide, using tiotropium bromide and ipratropium bromide as comparators. All four antagonists had high affinity for the five muscarinic receptor sub-types (M1-M5); aclidinium had comparable affinity to tiotropium but higher affinity than glycopyrronium and ipratropium for all receptors. Glycopyrronium dissociated faster from recombinant M3 receptors than aclidinium and tiotropium but more slowly than ipratropium; all four compounds dissociated more rapidly from M2 receptors than from M3 receptors. In vitro, aclidinium, glycopyrronium and tiotropium had a long duration of action at native M3 receptors (>8 h versus 42 min for ipratropium). In vivo, all compounds were equi-potent at reversing acetylcholine-induced bronchoconstriction. Aclidinium, glycopyrronium and ipratropium had a faster onset of bronchodilator action than tiotropium. Aclidinium had a longer duration of action than glycopyronnium (time to 50% recovery of effect [t½ offset] = 29 h and 13 h, respectively); these compare with a t½ offset of 64 h and 8 h for tiotropium and ipratropium, respectively. Aclidinium was less potent than glycopyrronium and tiotropium at inhibiting salivation in conscious rats (dose required to produce half-maximal effect [ED50] = 38, 0.74 and 0.88 µg/kg, respectively) and was more rapidly hydrolysed in rat, guinea pig and human plasma compared with glycopyrronium or tiotropium. These results indicate that while aclidinium and glycopyrronium are both potent antagonists at muscarinic receptors with similar kinetic selectivity for M3 receptors versus M2, aclidinium has a longer dissociation half-life at M3 receptors and a longer duration of bronchodilator action in vivo than glycopyrronium. The rapid plasma hydrolysis of aclidinium, coupled to its kinetic selectivity, may confer a reduced propensity for systemic anticholinergic side effects with aclidinium versus glycopyrronium and tiotropium.

Effects of aclidinium bromide in a cigarette smoke-exposed Guinea pig model of chronic obstructive pulmonary disease.

Long-acting muscarinic antagonists are widely used to treat chronic obstructive pulmonary disease (COPD). In addition to bronchodilation, muscarinic antagonism may affect pulmonary histopathological changes. The effects of long-acting muscarinic antagonists have not been thoroughly evaluated in experimental models of COPD induced by chronic exposure to cigarette smoke (CS). We investigated the effects of aclidinium bromide on pulmonary function, airway remodeling, and lung inflammation in a CS-exposed model of COPD. A total of 36 guinea pigs were exposed to CS and 22 were sham exposed for 24 weeks. Animals were nebulized daily with vehicle, 10 µg/ml, or 30 µg/ml aclidinium, resulting in six experimental groups. Pulmonary function was assessed weekly by whole-body plethysmography, determining the enhanced pause (Penh) at baseline, after treatment, and after CS/sham exposure. Lung changes were evaluated by morphometry and immunohistochemistry. CS exposure increased Penh in all conditions. CS-exposed animals treated with aclidinium showed lower baseline Penh than untreated animals (P = 0.02). CS induced thickening of all bronchial wall layers, airspace enlargement, and inflammatory cell infiltrate in airways and septa. Treatment with aclidinium abrogated the CS-induced smooth muscle enlargement in small airways (P = 0.001), and tended to reduce airspace enlargement (P = 0.054). Aclidinium also attenuated CS-induced neutrophilia in alveolar septa (P = 0.04). We conclude that, in guinea pigs chronically exposed to CS, aclidinium has an antiremodeling effect on small airways, which is associated with improved respiratory function, and attenuates neutrophilic infiltration in alveolar septa. These results indicate that, in COPD, aclidinium may exert beneficial effects on lung structure in addition to its bronchodilator action.

Phosphodiesterase-4 inhibitors: a review of current developments (2010 - 2012).

INTRODUCTION: At last, after many years of research, roflumilast has become the first oral phosphodiesterase-4 inhibitor to be approved by the medical agencies as an add-on therapy for chronic obstructive pulmonary disease. A second compound, apremilast is targeted for submission of new drug application for the treatment of psoriasis in the second half of 2013. These compounds represent a breakthrough and a reward in the field after the many failures to date in clinical development. AREAS COVERED: This review summarizes the clinical development of PDE4 inhibitors from 2010 - 2012 and the associated patent literature with a focus on strategies to overcome the common pitfalls of oral PDE4 inhibitors. EXPERT OPINION: In the last few years, influenced by the body of published clinical data, many companies have lost interest in PDE4 as a target. Many of those that have persevered have opted to realign their research programs either toward compounds specifically designed for inhaled delivery or in search of an increase in clinical efficacy by combining two mechanisms in a single compound. This change is reflected by the continued disclosure of novel and chemically diverse molecules, indicating for some in the pharmaceutical industry that all is not yet lost.

Aclidinium inhibits cigarette smoke-induced lung fibroblast-to-myofibroblast transition.

Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of this remodelling, peribronchiolar fibrosis is observed in the small airways of COPD patients and contributes to airway obstruction. Fibroblast-to-myofibroblast transition is a key step in peribronchiolar fibrosis formation. This in vitro study examined the effect of cigarette smoke on bronchial fibroblast-to-myofibroblast transition, and whether aclidinium bromide inhibits this process. Human bronchial fibroblasts were incubated with aclidinium bromide (10(-9)-10(-7) M) and exposed to cigarette smoke extract. Collagen type I and α-smooth muscle actin (α-SMA) expression were measured by real-time PCR and Western blotting, as myofibroblast markers. Intracellular reactive oxygen species, cyclic AMP (cAMP), extracellular signal-regulated kinase (ERK)1/2 and choline acetyltransferase were measured as intracellular signalling mediators. Cigarette smoke-induced collagen type I and α-SMA was mediated by the production of reactive oxygen species, the depletion of intracellular cAMP and the increase of ERK1/2 phosphorylation and choline acetyltransferase. These effects could be reversed by treatment with the anticholinergic aclidinium bromide, by silencing the mRNA of muscarinic receptors M1, M2 or M3, or by the depletion of extracellular acetylcholine by treatment with acetylcholinesterase. A non-neuronal cholinergic system is implicated in cigarette smoke-induced bronchial fibroblast-to-myofibroblast transition, which is inhibited by aclidinium bromide.

Pharmacological characterization of abediterol, a novel inhaled ß(2)-adrenoceptor agonist with long duration of action and a favorable safety profile in preclinical models.

Abediterol is a novel potent, long-acting inhaled ß(2)-adrenoceptor agonist in development for the treatment of asthma and chronic obstructive pulmonary disease. Abediterol shows subnanomolar affinity for the human ß(2)-adrenoceptor and a functional selectivity over ß(1)-adrenoceptors higher than that of formoterol and indacaterol in both a cellular model with overexpressed human receptors and isolated guinea pig tissue. Abediterol is a full agonist at the human ß(2)-adrenoceptor (E(max) = 91 ± 5% of the maximal effect of isoprenaline). The potency and onset of action that abediterol shows in isolated human bronchi (EC(50) = 1.9 ± 0.4 nM; t½ onset = 7-10 min) is not significantly different from that of formoterol, but its duration of action (t½ ∼ 690 min) is similar to that of indacaterol. Nebulized abediterol inhibits acetylcholine-induced bronchoconstriction in guinea pigs in a concentration-dependent manner, with higher potency and longer duration of action (t½ = 36 h) than salmeterol (t½ = 6 h) and formoterol (t½ = 4 h) and similar duration of action to indacaterol up to 48 h. In dogs, the bronchoprotective effect of abediterol is more sustained than that of salmeterol and indacaterol at doses without effects on heart rate, thus showing a greater safety margin (defined as the ratio of dose increasing heart rate by 5% and dose inhibiting bronchospasm by 50%) than salmeterol, formoterol, and indacaterol (5.6 versus 3.3, 2.2, and 0.3, respectively). In conclusion, our results suggest that abediterol has a preclinical profile for once-daily dosing in humans together with a fast onset of action and a favorable cardiovascular safety profile.

Aclidinium bromide, a novel long-acting muscarinic antagonist for COPD with improved preclinical renal and urinary safety profile.

AIMS: Aclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium. MAIN METHODS: The effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10-1000 µg/kg, s.c.), anaesthetised Beagle dogs (1000 µg/kg, i.v.) and anaesthetised guinea pigs (3-100µg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only). KEY FINDINGS: Aclidinium 1000 µg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 µg/kg which significantly decreased this parameter (p<0.05). Aclidinium 1000 µg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3-100 µg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 µg/kg decreased the peak micturition pressure (p<0.05), increased the volume of urine retained in the bladder (p<0.01) and showed a trend to decrease the volume of urine excreted. SIGNIFICANCE: Aclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.

Aclidinium inhibits human lung fibroblast to myofibroblast transition.

BACKGROUND: Fibroblast to myofibroblast transition is believed to contribute to airway remodelling in lung diseases such as asthma and chronic obstructive pulmonary disease. This study examines the role of aclidinium, a new long-acting muscarinic antagonist, on human fibroblast to myofibroblast transition. METHODS: Human bronchial fibroblasts were stimulated with carbachol (10(-8) to 10(-5) M) or transforming growth factor-ß1 (TGF-ß1; 2 ng/ml) in the presence or absence of aclidinium (10(-9) to 10(-7) M) or different drug modulators for 48 h. Characterisation of myofibroblasts was performed by analysis of collagen type I and α-smooth muscle actin (α-SMA) mRNA and protein expression as well as α-SMA microfilament immunofluorescence. ERK1/2 phosphorylation, RhoA-GTP and muscarinic receptors (M) 1, 2 and 3 protein expression were determined by western blot analysis and adenosine 3'-5' cyclic monophosphate levels were determined by ELISA. Proliferation and migration of fibroblasts were also assessed. RESULTS: Collagen type I and α-SMA mRNA and protein expression, as well as percentage α-SMA microfilament-positive cells, were upregulated in a similar way by carbachol and TGF-ß1, and aclidinium reversed these effects. Carbachol-induced myofibroblast transition was mediated by an increase in ERK1/2 phosphorylation, RhoA-GTP activation and cyclic monophosphate downregulation as well as by the autocrine TGF-ß1 release, which were effectively reduced by aclidinium. TGF-ß1 activated the non-neuronal cholinergic system. Suppression of M1, M2 or M3 partially prevented carbachol- and TGF-ß1-induced myofibroblast transition. Aclidinium dose-dependently reduced fibroblast proliferation and migration. CONCLUSION: Aclidinium inhibits human lung fibroblast to myofibrobast transition.